Immunoglobulins, such as antibodies, are of continued and increasing interest for the pharmaceutical industry. Since 2000, the therapeutic market for monoclonal antibodies has grown exponentially and in 2007, eight of the 20 best-selling biotechnology drugs in the U.S. were therapeutic monoclonal antibodies each having world wide annual sales of more than 5 billion USD.
Currently, a significant number of antibodies and also derivatives and fragments of immunoglobulins are in pre-clinical and clinical development. Before entry into humans, the drug under investigation has to be analysed and characterized in extensive discovery and pre-clinical testing. Important criteria like toxicological, pharmacokinetic and pharmacodynamic characteristics need to be explored for the establishment of a safe and potent drug profile. In order to quantify and monitor the therapeutic antibody levels, many of these studies require the use of drug-specific agents for the specific detection of the therapeutic antibody in a sample matrix like e.g. the sera or any body liquid from a patient or an experimental animal.
Drug-specific agents include e.g. antibodies that only detect human or humanized immunoglobulin and can therefore be used to quantify a human or humanized therapeutic antibody in a sample derived from a non-human experimental host (see e.g. WO2006066912; U.S. Ser. No. 11/792,910 which is incorporated by reference in its entirety). One step further is the use of anti-idiotypic antibodies or antibody fragments, which are specific for a unique structure within the therapeutic antibody. Therefore, anti-idiotypic antibodies can be used to detect a specific therapeutic antibody or antibody fragment in a sample matrix irrespective of the host the sample is isolated from (see e.g. WO2009032128). However, because the vast majority of anti-idiotypic antibodies bind to one or more of the unique CDRs of the therapeutic antibody and the CDRs define the paratope that specifically interacts with the antigen of the therapeutic antibody, only the detection and monitoring of free, non antigen-bound therapeutic antibodies are possible.
US 2012/0157663 describes so-called “domino antibodies” which have the capacity to bind to an antibody only if the antibody is bound to the respective antigen. The antibodies of US 2012/0157663 are generated via a specific hybridoma-based screening technology. Common to all domino antibodies is that the epitope of the domino antibody on the target antibody is formed through a conformational change upon binding of the target antibody to its respective antigen. The epitope is located in the constant region of the target antibody (e.g. the constant region of the light chain) and does not include any parts of the antigen nor the CDR region of the target antibody. In contrast, the complex-specific antibodies and antibody fragments of the present invention bind to at least certain parts of the CDR regions of the target antibody. Therefore, although domino antibodies only recognize target antibodies when the target antibodies are bound to their respective antigen, domino antibodies also bind to other target antibodies with the same antigen specificity, i.e. they are pan-specific in this respect. In contrast, the antibodies and antibody fragments of the present invention are specific for one single target antibody, and they only bind to this target antibody when the target antibody has bound to its antigen.
Since a therapeutic antibody that was applied to a patient is always balanced between different states within the periphery of the host's body, the monitoring and proportion of these different states provides mandatory information for the safety of the therapeutic antibody. These different states are balanced according to the law of mass action and comprise total antibody, unbound antibody and bound antibody and said balance is dependent e.g. on the affinity of the therapeutic antibody and also the concentration of the antigen in the body. Furthermore, due to the relatively slow clearance of therapeutic antibodies from the body, the therapeutic antibody bound to its antigen often leads to an increase of antigen levels upon its administration for a longer term (Charles P. (1999) Journal of Immunology 163; 1521-1528). In the presence of the therapeutic antibody the bound antigen is neutralized and predominantly is not bioactive. However, this phenomenon must be monitored and is important e.g. to assess the risk of an abrupt withdrawal of the drug.
Taken together the specific detection of total antibody, unbound antibody and bound antibody is of particular interest and importance for the profiling and later approval of a therapeutic antibody (Kuang B. (2010) Bioanalysis, 2(6):1125-40). Only a few anti-idiotypic antibodies are exemplified which are able to bind to the unbound therapeutic antibody and are also able to bind to the complex (therapeutic antibody bound to its antigen) and therefore are useful to detect the total antibody load. Such a non-paratopic anti-idiotypic antibody is disclosed in WO2009032128.
In contrast, almost all anti-idiotypic antibodies are directed to the CDRs of the target antibody and therefore only detect unbound antibody (see e.g. Tornetta M. (2007) Journal of Immunological Methods 328, 34-44).
However, neither the use of the CDR-specific anti-idiotypic antibody nor the use of the non-paratopic anti-idiotypic antibody enables the direct detection and quantification of the drug-antigen complex only. In order to quantify the bound antibody, various ELISA-based assays are established but always require the use of secondary, e.g. anti-human Fc, antibodies for indirect detection. The use of Fc-specific detection antibodies requires an extra step and extensive washing to capture and isolate the complex from the sera immunoglobulin and therefore these assays are susceptible for background noise and signal variations.
Accordingly alternative more sensitive and robust approaches are needed to detect and quantify antibody-antigen complexes.